Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo

Nat Protoc. 2009;4(12):1798-806. doi: 10.1038/nprot.2009.191.


Normal cells can permanently lose the ability to proliferate when challenged by potentially oncogenic stress, a process termed cellular senescence. Senescence-associated beta-galactosidase (SA-betagal) activity, detectable at pH 6.0, permits the identification of senescent cells in culture and mammalian tissues. Here we describe first a cytochemical protocol suitable for the histochemical detection of individual senescent cells both in culture and tissue biopsies. The second method is based on the alkalinization of lysosomes, followed by the use of 5-dodecanoylaminofluorescein di-beta-D-galactopyranoside (C12FDG), a fluorogenic substrate for betagal activity. The cytochemical method takes about 30 min to execute, and several hours to a day to develop and score. The fluorescence methods take between 4 and 8 h to execute and can be scored in a single day. The cytochemical method is applicable to tissue sections and requires simple reagents and equipment. The fluorescence-based methods have the advantages of being more quantitative and sensitive.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biomarkers / metabolism
  • Cellular Senescence*
  • Fluoresceins / analysis
  • Fluoresceins / chemistry
  • Galactosides / analysis
  • Galactosides / chemistry
  • Histocytochemistry / methods*
  • Hydrogen-Ion Concentration
  • Lysosomes / chemistry
  • Lysosomes / metabolism
  • Microscopy, Fluorescence
  • beta-Galactosidase / metabolism*


  • 5-dodecanoylaminofluorescein
  • Biomarkers
  • Fluoresceins
  • Galactosides
  • fluorescein-digalactoside
  • beta-Galactosidase